Struction And Expression Of The Growth Related Gene In Rongjiang Pig Of China

The growth and development of mammals are generally controlled by GH-IGF-I. We take the growth hormone gene and Insulin-like growth factor I as the major genes about the speed of animals, and clone and analyze the data which are related with the growth and development.The attempt has been made to better understand the growth and development of Rongjiang little pig and the relationship between growing speed and expression of IGF- I genes and growth hormone genes in Rongjiang little pig.Growth of animal is largely regulated by growth hormone (GH). In this study, the GH gene was isolated and cloned from the genomic DNA library from Rongjiang pig, a Chinese local swine, using polymerase chain reaction technique. The complete nucleotide sequence of a 1.903 Kb genomic fragment containing Rongjiang swine GH gene has been determined. The GH gene contained five exons and four introns similar to the GH genes of other mammalians and exhibited 97-99% identity to the GH genes of the four western meat-type breeds and nine Chinese local pigs. Polymorphism of GH genes was analyzed with the restriction enzymes Dde I, Nar I and BsmN I in four western meat-type breeds and ten Chinese local pigs. Five polymorphic restriction sites, with Dde I at the base 622(G/A) in exon2 and 274 (T/C) in 5'-flank, with Nar I at 631 (G/A) in exon2, and with BsmN I at the base 841 (T/C) in intron2 and 1358 (A/G) in exon4, were identified. The polymorphic restriction site at 1358 (A/G) leaded to the GH mature protein of Rongjiang pig differs from that of four western meat-type breeds and eight Chinese local breeds at the residue Val108 substituted by Ile108. According to the crystal structure of human GH mature protein, this Ile108 substitution might result in a lower affinity between GH and its receptor in Rongjiang breed.In order to detect the affinity between GH and its receptor in Rongjiang breed, we expressed and pured the mature peptide of pGH. It will provide the bases on materials and theory for deeply invest on application of pGH. We separated pGH gene from Rongjiang little pig pituitary issue using the RT-PCR technique.Specific pGH cDNA. 700bp, was amplified by RT-PCR with genomic RNA of pGH strain as the template for cDNA synthesis. PCR products were cloned into pMD18-T vector and named as pMD18-T-pGH. A 573bp-band was got by PCR with template of pMD18-T-fGH, after digested by restrict enzyme Sap I and Xho I. Then we ligated it into prokaryotic expressed plasmid of pTYB11 cut by Sap I and Xho I.We constructed prokaryotic expressed plasmid of pTYB11-pGH and transferred into E.coil ER2566. Then the mature protein was got successfully expressed. Under the inducement of final concentration 1mmol/L IPTG, the high expressed amount of GH protein was detected by SDS-PAGE electrophoresis and it occupied total E.coil protein 35%. The recombinant protein in Ecoil ER2566 was expressed in the form of excuse body. Cloning of Rongjiang little pig GH gene and the expression in prokaryotic system lay a foundation of farther study on the structure, biological function even operated machism of pGH gene on the level of cell and molecule. In this way, we can make a deep research on the practice aspect of promoting growth in mammalian GH gene. Insulin-like growth factors (IGFs) are consisted of two kinds of peptide, one is insulin-like growth factor I (IGF- I) and another is insulin-like growth factor II (IGF- II). Many studies showed that IGFs play very important regulated roles in the cell proliferation and apoptosis , pathogenesis of human cancer and tissue differentiation.Genes of IGFs, which include CDS, were cloned by way of RT-PCR from the tissue of human liver, then ligated the genes of IGFs to the vector of pMD18-T, the sequences of IGFs are correct by sequencing. Semi-quantitative RT-PCR was used to investigate the tissue expression of IGF-I gene in Rongjiang little pig. IGF-I was generally generated in the tissue of liver. The results showed that IGF-I expressed in liver, spleen, ovary, fattiness, cartilage and muscle. But not found in spinal and lung. The level of IGF- I mRNA in the liver was obviously higher than that in the other tissues. And we blast the IGF- I gene sequence in NCBI with that of other species, and also we can deduce the structure of IGF-I mature peptide, which can help to do a deep research on the practice aspect of promoting growth in mammalian GH gene.